Differential Effects of Sphingomyelin Hydrolysis and Cholesterol Transport on Oxysterol-binding Protein Phosphorylation and Golgi Localization

Protein kinase D (PKD) plays a critical role at the trans-Golgi network by regulating the fission of transport carriers destined for the plasma membrane. Two known Golgi-localized PKD substrates, PI4-kinase IIIβ and the ceramide transfer protein CERT, mediate PKD signaling to influence vesicle trafficking to the plasma membrane and sphingomyelin synthesis, respectively. PKD is recruited and activated at the Golgi through interaction with diacylglycerol, a pool of which is generated as a by-product of sphingomyelin synthesis from ceramide. Here we identify a novel substrate of PKD at the Golgi, the oxysterol-binding protein OSBP. Using a substrate-directed phospho-specific antibody that recognizes the optimal PKD consensus motif, we show that PKD phosphorylates OSBP at Ser240 in vitro and in cells. We further show that OSBP phosphorylation occurs at the Golgi. Phosphorylation of OSBP by PKD does not modulate dimerization, sterol binding, or affinity for PI(4)P. Instead, phosphorylation attenuates OSBP Golgi localization in response to 25-hydroxycholesterol and cholesterol depletion, impairs CERT Golgi localization, and promotes Golgi fragmentation.

Download Full-text

Golgi localization of oxysterol binding protein-related protein 4L (ORP4L) is regulated by ligand binding

Journal of Cell Science ◽

10.1242/jcs.215335 ◽

2018 ◽

Vol 131 (14) ◽

pp. jcs215335 ◽

Cited By ~ 3

Author(s):

Antonietta Pietrangelo ◽

Neale D. Ridgway

Keyword(s):

Ligand Binding ◽

Binding Protein ◽

Related Protein ◽

Oxysterol Binding Protein ◽

Golgi Localization

Download Full-text

Golgi localization and phosphorylation of oxysterol binding protein in Niemann-Pick C and U18666A-treated cells

The Journal of Lipid Research ◽

10.1016/s0022-2275(20)31595-9 ◽

2001 ◽

Vol 42 (7) ◽

pp. 1062-1071 ◽

Cited By ~ 1

Author(s):

Abbas Mohammadi ◽

Ryan J. Perry ◽

Margo K. Storey ◽

Harold W. Cook ◽

David M. Byers ◽

...

Keyword(s):

Binding Protein ◽

Oxysterol Binding Protein ◽

Golgi Localization ◽

Niemann Pick

Download Full-text

Oxysterol-binding protein-related protein 1 variants have opposing cholesterol transport activities from the endolysosomes

Molecular Biology of the Cell ◽

10.1091/mbc.e19-12-0697 ◽

2020 ◽

Vol 31 (8) ◽

pp. 793-802 ◽

Cited By ~ 4

Author(s):

Kexin Zhao ◽

Jason Foster ◽

Neale D. Ridgway

Keyword(s):

Plasma Membrane ◽

Cholesterol Efflux ◽

Binding Protein ◽

Ldl Receptor ◽

Full Length ◽

Receptor Activity ◽

Cholesterol Transport ◽

Related Protein ◽

Oxysterol Binding Protein ◽

Late Endosomes

OSBPL1 encodes the full-length ORP1L and the truncated variant ORP1S. ORP1S is responsible for transferring cholesterol from late endosomes to the plasma membrane to regulate cholesterol efflux by ABCA1 and LDL receptor activity. ORP1L and ORP1S combine to transport cholesterol from late endosomes to the ER and PM, respectively.

Download Full-text

Oxysterol-binding protein (OSBP) is required for the perinuclear localization of intra-Golgi v-SNAREs

Molecular Biology of the Cell ◽

10.1091/mbc.e13-05-0250 ◽

2013 ◽

Vol 24 (22) ◽

pp. 3534-3544 ◽

Cited By ~ 13

Author(s):

Taki Nishimura ◽

Yasunori Uchida ◽

Rieko Yachi ◽

Tetyana Kudlyk ◽

Vladimir Lupashin ◽

...

Keyword(s):

Cholesterol Level ◽

Binding Protein ◽

Cholesterol Depletion ◽

Oxysterol Binding Protein ◽

Golgi Localization ◽

Protein Receptors ◽

Intracellular Organelles ◽

Endoplasmic Reticulum Targeting ◽

Related Proteins ◽

Interfering Rna

Oxysterol-binding protein (OSBP) and OSBP-related proteins (ORPs) have been implicated in the distribution of sterols among intracellular organelles. OSBP regulates the Golgi cholesterol level, but how it relates to Golgi function is elusive. Here we report that OSBP is essential for the localization of intra-Golgi soluble vesicle N-ethylmaleimide-sensitive fusion attachment protein receptors (v-SNAREs). Depletion of OSBP by small interfering RNA causes mislocalization of intra-Golgi v-SNAREs GS28 and GS15 throughout the cytoplasm without affecting the perinuclear localization of Golgi target-SNARE syntaxin5 and reduces the abundance of a Golgi enzyme, mannosidase II (Man II). GS28 mislocalization and Man II reduction are also induced by cellular cholesterol depletion. Three domains of OSBP—an endoplasmic reticulum–targeting domain, a Golgi-targeting domain, and a sterol-binding domain—are all required for Golgi localization of GS28. Finally, GS28 mislocalization and Man II reduction in OSBP-depleted cells are largely restored by depletion of ArfGAP1, a regulator of the budding of coat protein complex (COP)-I vesicles. From these results, we postulate that Golgi cholesterol level, which is controlled by OSBP, is essential for Golgi localization of intra-Golgi v-SNAREs by ensuring proper COP-I vesicle transport.

Download Full-text

Oxysterol-binding-protein (OSBP)-related protein 4 binds25-hydroxycholesterol and interacts with vimentin intermediate filaments

Biochemical Journal ◽

10.1042/bj3610461 ◽

2002 ◽

Vol 361 (3) ◽

pp. 461-472 ◽

Cited By ~ 55

Author(s):

Cheng WANG ◽

Lellean JeBAILEY ◽

Neale D. RIDGWAY

Keyword(s):

Intermediate Filaments ◽

Intermediate Filament ◽

Cellular Localization ◽

Cholesterol Metabolism ◽

Binding Protein ◽

Cholesterol Transport ◽

Ph Domain ◽

Transport Pathway ◽

Oxysterol Binding Protein ◽

Filament Network

Oxysterol-binding protein (OSBP) is the prototypical member of a class of phospholipid and oxysterol-binding proteins that interacts with the Golgi apparatus and regulates lipid and cholesterol metabolism. As a result of recent sequencing efforts, eleven other OSBP-related proteins (ORPs) have been identified in humans. We have investigated the structure, oxysterol-binding activity, cellular localization and function of ORP4 (also designated OSBP2 or HLM), a homologue that shares the highest degree of similarity with OSBP. Two ORP4 cDNAs were identified: a full-length ORP4 containing a pleckstrin homology (PH) domain and an oxysterol-binding region (designated ORP4-L), and a splice variant in which the PH domain and part of the oxysterol-binding domain were deleted (designated ORP4-S). ORP4 mRNA and protein expression overlapped partially with OSBP and were restricted to brain, heart, muscle and kidney. Like OSBP, ORP4-L bound [3H]25-hydroxycholesterol with high affinity and specificity. In contrast, ORP4-S did not bind [3H]25-hydroxycholesterol or [3H]7-ketocholesterol. Immunofluorescence localization in stably transfected Chinese hamster ovary cells showed that ORP4-S co-localized with vimentin and caused the intermediate filament network to bundle or aggregate. ORP4-L displayed a diffuse staining pattern that did not overlap with vimentin except when the microtubule network was disrupted with nocodazole. Oxysterols had no effect on the localization of either ORP4. Cells overexpressing ORP4-S had a 40% reduction in the esterification of low-density-lipoprotein-derived cholesterol, demonstrating that ORP4 interaction with intermediate filaments inhibits an intracellular cholesterol-transport pathway mediated by vimentin. These studies elucidate a hitherto unknown relationship between OSBPs and the intermediate filament network that influences cholesterol transport.

Download Full-text